Pak. J. Bot., 52(4): 1305-1313, 2020. DOI: http://dx.doi.org/10.30848/PJB2020-4(34)
DIFFERENTIAL EXPRESSION OF INDOLE ALKALOID PATHWAY GENES ACROSS
CATHARANTHUS ROSEUS PLANT ORGANS
AHMED M. RAMADAN1,2*, MUNA A. ABDULGADER1, THANA KHAN1,
NOUR O. GADALLA3,4 AND AHMED BAHIELDIN1,5
1Department of Biological Science, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
2Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, Egypt 3Department of Arid Land Agriculture, Faculty of Meteorology, Environment and Arid Land Agriculture,
King Abdulaziz University, Jeddah, Saudi Arabia 4Genetics and Cytology Department, Genetic Engineering and Biotechnology Division,
National Research Center, Dokki, Egypt 5Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt
*Corresponding author’s email: [email protected]
Abstract
Vinblastine and vincristine are two indole alkaloids of the medicinal plant Catharanthus roseus, widely used in cancer
chemotherapy such as leukemia, Hodgkin's disease and solid tumors. Transcriptomic data of C. roseus across different organs (stem, hairy root, flower, immature and mature leaves) was collected and de novo assembled then differential expression analysis was achieved. The target of this study was the detection of components of terpenoid indole alkaloid pathway based on gene expression analysis. The in-silico analysis was focused on six genes (SLS, sgd, STR1, OMT, DAT and G8H) of the TIA pathway that were expressed in different plant organs. The results indicated posability of formed enzyme in organ and transfer to other organs to work and restore the TIA pathway. The highest expressions level of most TIA key genes took places in root, flowers and mature leaves.
Key words: Transcriptome analysis, vinblastine, vincristine, de novo, in silico.
Introduction
Plants are attractive sources of pharmaceutical compounds used for therapeutic purposes, especially anti-cancer compounds, albeit being naturally synthesized in plants for the defence against pests and diseases (Ziegler & Facchini, 2008; Roepke et al., 2010; Perveen et al., 2020). Several other important pharmaceutical compounds exist in higher plants such as baccharin, ellipticine, homoharringtonine, tripdiolide, bruceantin, indicine-N-oxide, thalicarpine and maytansine (Ziegler & Facchini, 2008; Guirimand et al., 2010). Among plant products that are marketed as anti-cancer drugs are vinblastine, vincristine, etoposide and camptothecin. Some of these pharmaceutical compounds, e.g., vinblastine and vincristine, are produced in terpenoid indole alkaloid (TIA) pathway of Catharanthus roseus and are widely used in cancer chemotherapy against various leukemia, Hodgkin's disease, solid tumors, and depression (Contin et al., 1998; Dogru et al., 2000; Lange et al., 2001; Burlat et al., 2004; Hamid et al., 2017).
Apocynaceae is a family of medicinal plants including
about 550 genera that are considered as a rich resource of
TIA. Catharanthus roseus is a member of this family and
comprises about 130 TIA; 25 of them are dimeric indole
alkaloids. TIA biosynthesis pathway has received
considerable attention; however, the full characterization of
alkaloids pathways is not completely accomplished (Zhu et
al., 2015). TIAs are metabolites that have antitumor features
such as vincristine and vinblastine that are utilized to treat
many types of cancer (Sofowora et al., 2013; Greenwell and
Rahman, 2015; Nejat et al., 2015; Soares et al., 2016). The
general mechanisms of these metabolites are destroying
cancer cells through inhibiting tubulin polymerization; and
attacking enzymes involved in DNA duplication and
transcription such as topoisomerases I and II (Chi et al.,
2015; Chan et al., 2015; Montecucco et al., 2015).
Pharmaceutical compounds synthesized in C. roseus
plant are extracted at low concentrations. The
concentrations of vinblastine and vincristine did not exceed
0.0005% of the dry matter (Neumann et al., 2009). Earlier
efforts to produce these useful compounds more efficiently
relied on plant tissue culture and recombinant DNA
technologies (Carew, 1966). Recently, several studies have
used next-generation sequencing (NGS) tools to get a new
vision on the eukaryotic transcriptome size and complexity
(Wang et al., 2009). RNA-Seq has emerged as a powerful
platform for providing an enormous and accurate millions
of transcriptome sequence data. Researchers utilized NGS
to illustrate the function of novel genes at a high-
throughput basis (Oliver, 2002; Yegnasubramanian, 2013).
The main advantages of RNA data analysis are the
accuracy, low cost, high coverage, and great resolution
compared to traditional methods (Kukurba & Montgomery,
2015; Park et al., 2016). RNA-seq analysis of gene
expression have played a critical role in defining TIA
encoded genes in C. roseus (El-Domyatiet al., 2017). This
study was designed to detect spatial and temporal
transcriptome profiling in C. roseus with emphasis on TIA
pathway across different organs.
Materials and Methods
RNA-Seq data collection: Transcriptome sequences of
C. roseus (Little Bright Eyes cultivar) were obtained from
SRA (NCBI) with accession numbers of SRR122239
(flower), SRR122243 (immature leaf), SRR122251
(mature leaf), SRR122253 (stem), SRR122254 (root), and
SRR122257 (hairy root). Data quality was measured
using fastQC program after trimming adapter sequences.
Read length of ≤ 50 bp and low qualities reads (≥ 20 for
all bases) were removed.
AHMED M. RAMADAN ET AL., 1306
Sequence assembly, genes annotation and clusters
analysis: Recovered RNA-Seq reads were de novo
assembled at kmer value = 50 and analyzed using Trinity
RNA-Seq assembly (version r2013-2-25) and the tuxedo
suite software packages. The relative abundance of the
resulted transcripts was estimated using RSEM (v1.1.6).
For differential expression analysis, gene clusters formed
for each organ were analyzed via EdgeR software (version
3.0.0, R version 2.1.5). Blast-2-GO software (version 2.3.5)
was used to detect differentially expressed (DE) transcripts.
Clusters with functional enrichment were identified by
setting a significant Pearson’s correlation through
permutation analysis (Brown et al., 2006).
Validation of RNA-Seq datasets: Biological samples
were collected from stem, hairy root, flower, root,
immature and mature leaves (three months old) of C.
roseus SunStorm™ cv. Apricot. Plants were grown at
25°C in growth chamber with 12 hours light as described
(Brenchley et al., 2012). RNAs were isolated using
RneasyTM kit and relative expression of the SLS, sgd,
STR1, OMT and DAT genes of C. roseus was detected via
qRT-PCR to validate the RNA-Seq data. To synthesize
cDNA, 2 µg of total RNA, oligo (dT) and SuperScript III
Reverse Transcriptase (Invitrogen™ cat. no. 18080044)
was used. Primer BLAST was used to design specific
primers (Table 1) for qRT-PCR. Templates were
amplified to recover 196 bp fragment of the C. roseus
actin gene (accession no. EF688556). qRT-PCR was done
according to Ramadan et al. (2019). Gene relative
expression to the internal reference control was calculated
using equation : 2-ΔΔCT = {(CT target gene – CT internal
control) tissue A-(CT target gene – CT internal control)
tissue B} (Thomas & Kenneth, 2008).
Table 1. Primer sequences along with the annealing temperatures and expected band sizes (bp) to be utilized in
validating RNA-Seq dataset of C. roseus via qRT-PCR. The actin gene was used as
the house-keeping gene (196 bp).
Gene Acc. no. Forward primer Reverse primer Anneal
temp.
SGD EU072423 AGCTCTTGTAGGAAGCCGTC CGTAACCCGGAGTATCGGGA 60 ºC
OMT EF444544 TCTACCGCCTAATGCGTGTT AAGTTGAACAGGGTCAGCCA 60 ºC
DAT AF053307 TGAAGGATTGGGCTGCTTCT TTCTATGGCTTCCGGAGGGA 58ºC
SLS KF309242 AGGACACAAAGTTAGGGCCG CTTGGTGGCATTGGCAACTC 58 ºC
STR1 Y10182 AGCGCAGATGGTTCCTTTGT ACCCAAAAATGGCCATCAGAA 60 ºC
Actin EF688556 TGGTCGTCCAAGACACACTG CTCTTCAGGGGCAACACGAA 60 ºC
Table 2. Statistics analysis of Catharanthus roseus RNA-Seq data. F=flower, IML=immature leaf, ML=mature leaf, S=stem,
R=root, HR=hairy root.
Transcriptome data
file (a)
Organ organs
(b)
Total no. of
reads (c)
No. of mapped
reads (d)
% of mapped
reads (e)
No. of
unmapped
reads (f)
% of unmapped
reads (g)
Total no. of
transcripts (h)
SRR122239.fastq Flower 31645190 28970625 91.55% 2674565 8.45% 40344
SRR122243.fastq Immature leaf 29918941 27580411 92.18% 2338530 7.82% 39952
SRR122251.fastq Mature leaf 25325632 23317495 92.07% 2008137 7.93% 40336
SRR122253.fastq Stem 28903929 26398362 91.33% 2505567 8.67% 40936
SRR122254.fastq Root 30896527 29222863 94.58% 1673664 5.42% 41281
SRR122257.fastq Hairy root 25316070 23404947 92.45% 1911123 7.55% 41806 aC. roseus transcriptome data files bThe organ of each transcriptome file cTotal number of reads recovered from C. roseus RNA-Seq data dNumber of de novo assembled mapped reads across each organ ePercentage of de novo assembled reads fNumber of unmapped reads gPercentage of unmapped reads hTotal number of transcripts generated from de novo assembled contigs
Results
Analysis of RNA-Seq datasets: RNA sequencing of C.
roseus transcriptome data yielded between ~ 25-31
million reads (Table 2). The number of de novo
assembled mapped reads was 158,894,703 comprising
percentages of ~91-94% across organs. The total number
of transcripts generated from de novo assembly was
244,655 transcripts comprising ~39,000-42,000 across
C. roseus organs.
Clusters of gene expression in different organs:
Differential expression (DE) of transcripts across C. roseus
organs was detected from RNA-Seq data. To confirm the
differences statistically, likelihood ratio was used to
compare RPKM-derived read counts with a threshold of ≥
2 RPKM value. Two-fold expression rate of transcripts
changes and 10-3 of false discovery rate were specified. The
number of deferentially expressed transcripts resulted from
de novo assembly was 2,266 transcripts. Pearson’s
DIFFERENTIAL EXPRESSION OF INDOLE ALKALOID PATHWAY GENES 1307
correlation was used to analyze clusters in order to sort out
expression profiles of DE transcripts and subject them to
hierarchical clustering (Fig. 1). They were sorted out based
on the relative abundance of transcripts across different
organs. The cluster analysis of gene expression levels
provided information for transcript expression patterns and
pathways across different organs. In general, the number of
upregulated clusters was 16 in immature leaf, while 13 in
flower, 12 in mature leaf, 6 in stem, 6 in root, and 4 in
hairy roots. Six out of these upregulated clusters (nos. 7,
22, 94, 97, 100 and 154; Fig. 2) contained the eight genes
of TIA pathway.
Fig. 1. Hierarchical cluster analysis of gene expression across different C. roseus organs.
AHMED M. RAMADAN ET AL., 1308
Fig. 2. Six selected clusters of DE transcripts from different C. roseus organs.
DIFFERENTIAL EXPRESSION OF INDOLE ALKALOID PATHWAY GENES 1309
Fig. 3. Indole Alkaloid biosynthesis in TIA Pathway (El- Domyati et al., 2017).
AHMED M. RAMADAN ET AL., 1310
Analysis of differentially expressed transcripts: According
to TIA pathway (Fig. 3), upregulation was found in the three
genes in the beginning of the TIA pathway in mature leaf,
flower and root (Fig. 4). These genes encode enzymes
secologanin synthase (SLS) (comp28933_c0), strictosidine
synthase (STR1) (comp11842_c0), and strictosidine beta-
glucosidase (SGD) (comp16844_c0) that exist in cluster 94.
In flower, the accumulation of transcripts encoding
tabersonine 16-O-methyltransferase (OMT),
deacetylvindoline_4-O-acetyltransferase (DAT),
hydroxytabersonine N-methyltransferase (NMT) and
peroxidase n1 (PRX1) seem not to support the accumulation
of vindoline, vincristine and vinblastine. Therefore, it is
suggested that the TIA pathway in flower is directed towards
the accumulation of other alkaloids such as ajmaline,
serpentine, raucaffricine or sarpagine. However, in mature
leaf, the accumulation of these transcripts was very high,
indicating the possible high accumulation of vincristine,
vinblastine, vindoline and possibly catharanthine. In hairy root, accumulation of previous transcripts was not observed,
which indicated that vincristine, vinblastine, vindoline
unlikely be accumulated in this organs. In stem,
accumulation of these genes does not support accumulation
of the latter alkaloids except for deacetylvindoline_4-O-
acetyltransferase (DAT) (Fig. 4). In immature leaf, high
accumulation of geraniol 8-hydroxylase (comp34847) was
observed with no accumulation of secologanin synthase
(SLS), indicating the possible accumulation of asperuloside
in immature leaf (Fig. 5).
RNA-Seq data Validation: To validate RNA-Seq data,
relative gene expression analysis was done via qRT-PCR.
The data showed the same expression patterns of the five
transcripts SLS, sgd, STR1, OMT and DAT in the RNA-
Seq datasets (Fig. 6).
Discussion
The biosynthesis of C. roseus terpenoid indole
alkaloid has been studied for decades. Although there is
tremendous progress, many steps are still not deciphered.
To date, C. roseus is the only natural resource for
antitumor agents, vinblastine and vincristine. However,
their contents in plants are very low. Furthermore, the
chemical synthesis of these components is far from being
applicable for commercial-scale production (Zhu et al.,
2015). Many investigators aimed to detect the
accumulation of indole alkaloids in different C. roseus
organs (Roepke et al., 2010; Zhu et al., 2015; Benyammi
et al., 2016). It was also reported that some indole
alkaloids like vindoline and catharanthine in a given
organ can be transferred to another organ. Accumulation
of these alkaloids in C. roseus mainly take place in young
leaves and stems, whereas synthesis of others such as
ajmalicine and serpentine mainly occurs in roots (Liu et
al., 2016). The main aims of this study are to understand
TIA biosynthetic pathways in C. roseus and detect the
organ of which genes involved in this process are
regulated. Transcriptome data of C. rosues collected from
NCBI indicates that DE transcripts in each cluster differ
in their expression levels across C. roseus organs. The
enzymes expressed from these DE transcripts act in
synthesizing TIA compounds. The six DE transcripts for
TIA biosynthesis in this study were subjected to Kyoto
Encyclopedia of Genes and Genomes (KEGG) analysis.
There are many reports that clarified that high
accumulations of vindoline, vinblastine and vincristine
are mainly in mature leaf whereas catharanthine was
highly accumulated in root (Malik et al., 2013; Srivastava
et al., 2014). Our findings supported the previous
investigation due to the expression profile of SLS, STR1,
sgd, OMT, DAT and PRX1 genes, but we found no
expression of NMT gene in mature leaf although it was
found in root and flower. Therefore, we suggest that the
NMT protein may be transferred from root to leaf as a
step towards the completion of the TIA pathway. The
transferred TIA proteins were proven in many reports
(Yamamoto et al., 2016). Also, our finding supports
previous investigation regarding the accumulation of
catharanthine in root due to the high expression of SLS,
STR and sgd genes in root, while no or low expression of
other TIA pathway genes was observed.
Another interesting finding was that G8H transcript
was highly accumulated in immature leaf. This outcome
suggests: (1) the accumulation of asperuloside TIA
product in immature leaves tissues due to the activity of
G8H enzyme and absence of SLS transcript activity and
(2) the transferring of G8H enzyme from immature leaves
to mature leaves tissues to open TIAs pathways in the
presence of SLS transcript activity.
Although we detected the possible location of
expression of some TIA gene in organs by transcriptome
analysis, indole alkaloids accumulation in plant organs is
still ambiguous as the accumulation of some indole
alkaloids in a given organ is not associated with the
location of gene expression or with the accumulation of
the encoded enzyme in the same organ. Rather, the
enzymes are likely transferred from one organ to the other
in order to complete the TIA pathway in the distal organ.
However, many steps of Tabrsonine accumulation are still
unknown.
Conclusions
Transcriptome analysis of C. roseus enabled us to
detect the expression levels of six genes related to TIA
biosynthesis pathway across different organs. Results
showed that posability of transfer of formed enzyme in
one organ and transfer to other organs to work and
restore the TIA pathway. the highest expressions level
of most TIA key genes took places in root, flower
and mature leaf. These findings may be useful in
future to determine the precise mechanism of TIA
biosynthesis in C. roseus organs as well as link
coexpressed genes and transcription factors involved
in this process. Furthermore, it is important to
investigate processes facilitating transport of enzymes
from one organ to the other.
DIFFERENTIAL EXPRESSION OF INDOLE ALKALOID PATHWAY GENES 1311
Fig. 4. Expression of transcripts related to the TIA biosynthetic pathway in C. roseus organs. SLS = secologanin synthase, STR1 =
strictosidine synthase1, sgd = strictosidine beta-glucosidase, PRX1= peroxidase 1, NMT = hydroxytabersonine N-methyltransferase,
OMT= tabersonine 16-O-methyltransferase, DAT = deacetylvindoline_4-O-acetyltransferase.
Fig. 5. Expression of geraniol 8-hydroxylase (G8H) transcripts related to the Monoterpenoid biosynthesis in C. roseus organs.
AHMED M. RAMADAN ET AL., 1312
Fig. 6. Relative gene expression analysis of SLS, sgd, STR1, OMT, and DAT transcripts of C. roseus in different organs.
Acknowledgments
This project was funded by the Deanship of scientific Research
(DSR) at King Abdulaziz University, Jeddah, under grant no. G-
184-130-38. The authors, therefore, acknowledge with thanks
DSR technical and financial support
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(Received for publication 20 November 2018)